Improving the aqueous solubility of reagents, preventing their aggregation, lowering their non-specific binding and reducing their non-specific interactions with sample components in serum or whole blood, remain ongoing challenges in clinical diagnostic assays. Commercial diagnostic immunoassays often use hydrophobic chemiluminescent and fluorescent labels that have limited aqueous solubility and these labels can exacerbate the non-specific binding of proteins, such as antibodies. Increasingly, recombinant proteins and peptides are being used in diagnostics and often these polypeptides exhibit poor aqueous solubility as well as a tendency to form insoluble aggregates because of misfolding or denaturation. Reagents can also interact with sample components in a non-specific manner giving rise to false positives in assays.
Several strategies to address some of the above mentioned issues have been described in the literature. For example, Basu et al. (Bioconjugate Chem. 2006, 17, 618-630) and Marsac et al. (Bioconjugate Chem. 2006, 17, 1492-1498) have described the use of poly(ethylene)glycol (PEG) to improve the solubility of polypeptides and proteins. Natrajan et al. (U.S. Pat. Nos. 6,664,032 and 7,309,615)) on the other hand have described the use of PEG to improve the aqueous solubility of hydrophobic, chemiluminescent, acridinium ester labels. Goldberg and coworkers (Biophysical Chem. 2003, 100, 569-479) synthesized a variety of non-detergent, zwitterionic sulfobetaines (NDSBs) and showed that these compounds are useful additives in assisting refolding of proteins such as BSA as well as enzymes and a monoclonal antibody. Similarly, D'Amico and Feller, (Anal. Biochem. 2009, 385, 389-391) have shown that the addition of an NDSB inhibited the thermal denaturation of several proteins. In another approach, Tolbert and coworkers (Bioconjugate Chem. 2008, 19, 1113-1118) have recently reported that site-specific modification of two aggregation-prone polypeptides with betaine (trimethylammonium moiety) significantly improved the aqueous solubility of the two polypeptides and inhibited aggregate formation.
Besides protein modification to improve their solubility, PEG has also been used devise inert surfaces that resist protein adsorption. For example, Ostuni et al. in J. Am. Chem. Soc. 2000, 17, 5605-5620, evaluated numerous functional groups attached to self-assembled monolayers for resistance to protein adsorption and observed that poly(ethylene)glycol functional groups conferred the best resistance. More recently, Jiang and coworkers in Biomacromolecules 2008, 9, 1357-1361; and others have reported that zwitterion-modified hydrophilic surfaces are as inert to protein adsorption as PEG. Structures of some zwitterions such as sulfobetaines, carboxybetaines, phosphobetaines and amine oxides are shown in the exemplary structures below. Like PEG, these zwitterions are normally electrically neutral, because of the balance of positive and negative charges within a given structure (R1-R4 are typically alkyl groups).